I am working with a protein-12kda; my vector contains 10X His tag
My protocol for purification-
NOTE- this is from 1 ml sonicated supernatant, used 100ul NiNTA beads.
After I collect Flow Through (FT), I always collect Beads-BB approx 10ul to see the binding capacity, Then I wash the column ( containing my beads) with Lysis Buffer (50mm TrisCl ph 7.5, 300mm NaCl, 10% Glycerol)
i.e. Lysis buffer without Imidazole
Then I wash the beads with Lysis Buffer + Different concentration of Imidazole (5mm, 10mm, 20mm to 300ml)
After I run SDS PAGE , I observed that my protein gets washed out in Lysis buffer without imidazole.
I have attached a Gel Picture –
Uninduced, Ladder, FT, Bead Binding BB, Lysis Buffer , Lysis buffer +Imidazole-5mm , 10mm and so on
Please help me to solve my problem.
NOTE - I tried with TrisCl Ph 8.0 and also with 500mm Nacl but I got the same result
Thank you.
Is the major band at the expected molecular weight of your protein?
Do you know that your protein is being expressed?
@adam
Sir , my protein is being expressed as I always get band n also after sonication I get my protein in soluble form too but when I go for purification I face this problem
If the protein is in an aggregated but soluble form, it may not bind well because the His tag is obscured.
If the protein undergoes proteolysis causing the His tag to be cleaved off, it will not bind.
Cut out the band from the gel and submit it to a protein sequencing facility to determine whether it is the right protein and whether it contains the His tag.
@xu zhang
I used 10%glycerol .. I will wash next time twice then.. and during binding I keep at rocker shaker at normal speed...not so high..not so low...
@xu zhang
Thank you
I will try without glycerol next time .... If u get any idea please let me know in future.
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